Studies on the genetic diversity of species with pharmacological potential are essential for the conservation of genetic resources. The genetic characterization of a species can guide efforts to collect and conserve germplasm and, in the future, genetic improvement. Domestic cultivation of medicinal plants is an important strategy for protecting these species from over harvesting. Genetic studies of medicinal species by molecular markers could allow for assessment and management of these species. Croton urucurana Baill. is widely used in Brazilian traditional medicine, but there are practically no data about its genetic patterns. We used 12 ISSR markers to evaluate the genetic diversity levels of 30 trees distributed in three populations of C. urucurana, two of which were located in Brazilian savanna (Cerrado) and one on semi-deciduous tropical forest areas. From a total of 82 bands recorded, 89% were polymorphic and presented a Shannon’s diversity index of 0.26. The populations formed two distinct groups: Two populations were divided each into a different genetic group, while the Aquidauana population, which is in a transition area between the Cerrado and the Pantanal, presented a mixture of the two genetic groups. Croton urucurana has G_ST values (0.200) similar to those found in species with a mixed mating system, but the lack of detailed studies on the reproductive system of this species makes it difficult to correctly interpret the origin of the population structure observed. Moreover, even using a simpler marker we were able to identify regions that deserve further studies, especially from a chemical point of view, to see whether there are any differences between the populations that could help in defining future conservation strategies, management and genetic improvement plans of the species.